JP6816026B2 - Cell proliferation - Google Patents

Description

This application claims the priority of U.S. Patent Provisional Application No. 62 / 161,128 (Title: Cell Proliferation) filed May 13, 2015, of the U.S. Patent Provisional Application. The whole is explicitly incorporated into this application by the disclosure herein.

The Cell Proliferation System (CES) is used to proliferate and differentiate various cells used for research and therapeutic purposes. As an example, T cell therapy (eg, autologous T cell therapy) is a new technique for treating many diseases. For example, in vitro genetically engineered and proliferated T cells are promising as a treatment for leukemia (eg, B cells) and other cancers.

Currently, T cell proliferation takes place within bag systems and systems, but such systems are not fully automated and are not functionally closed.

Embodiments of the present invention have been made in consideration of the above points and other points. However, the issues described herein do not limit the use of embodiments of the present invention.

This section is intended to illustrate aspects of some embodiments of the invention in a simple form and is not intended to identify the important or essential elements of the invention claimed. It is not intended to limit the scope of the claims.

Embodiments relate to a cell proliferation system (CES) and a method of growing cells in a cell proliferation system bioreactor. In an embodiment, cells are grown in a bioreactor such as a hollow fiber bioreactor. In an embodiment, cells (eg, T cells) are introduced into a hollow fiber bioreactor containing a plurality of hollow fibers. In an embodiment, the first plurality of cells are exposed to an activator to activate the proliferation of the cells in the hollow fiber bioreactor. In embodiments, the activator is a cell or (eg, on beads) bound antigen or soluble antigen. After exposing the cells to the activator, the cells are proliferated to produce a second plurality of proliferating cells. Then, the second proliferating cell is taken out from the bioreactor.

In other embodiments, a cell proliferation system for proliferating cells is provided. In an embodiment, the system comprises a hollow fiber bioreactor with a first fluid flow path having at least both ends. The first end of the first fluid flow path is fluidly associated with the first port of the hollow thread bioreactor, and the second end of the first fluid flow path is of the hollow thread bioreactor. Fluidly associated with the second port, the first fluid flow path has a capillary inner portion of the hollow filament bioreactor. A plurality of cells are introduced into the first fluid flow path using a fluid inlet path fluidly associated with the first fluid flow path. A pump that circulates the fluid in the first fluid flow path is further provided.

The system has a processor that executes instructions. When the instruction is executed by the processor, a method comprising the step of introducing a first fluid containing a first plurality of cells containing white blood cells into the inner part of the capillary of the hollow filament bioreactor is performed. The processor further exposes the first plurality of cells to the first activator in the medial portion of the capillary to activate the proliferation of the first plurality of cells in the hollow fiber bioreactor. Execute the command to be executed. The processor introduces a second fluid containing a medium into the inner portion of the capillary, proliferates at least a part of the first plurality of cells, and produces a second plurality of proliferating cells. Execute the command to do. The processor executes an instruction to perform a step of removing the second plurality of proliferating cells from the bioreactor.

Non-limiting and non-comprehensive embodiments are described with reference to the accompanying drawings.

FIG. 1 is a perspective view of a hollow fiber type bioreactor according to an embodiment of the present invention. FIG. 2 is a perspective view of a cell proliferation system provided with a pre-installed fluid transfer device according to an embodiment of the present invention. FIG. 3 is a perspective view of the housing of the cell proliferation system according to the embodiment of the present invention. FIG. 4 is a perspective view of a pre-installed fluid transfer device according to an embodiment of the present invention. FIG. 5 is a schematic view of a cell proliferation system according to an embodiment of the present invention. FIG. 6 is a schematic diagram of another embodiment of the cell proliferation system. FIG. 7 is a flowchart of the cell proliferation process according to the embodiment of the present invention. FIG. 8 is a diagram showing components of a computer system used to carry out embodiments of the present invention. FIG. 9 is a bar graph showing the total number of cells at various time points during culturing according to one embodiment. FIG. 10 is a bar graph showing cell viability at various time points during culturing according to one embodiment. FIG. 11 is a bar graph showing the number of dendritic cells with respect to the input of purified cells and the input of unpurified cells according to one embodiment. FIG. 12 is a bar graph showing the total number of cells at different time points during culturing (and for different purityes at cell input) according to one embodiment. FIG. 13 is a bar graph showing cell viability at different time points during culturing (and for different purityes at cell input) according to one embodiment. FIG. 14 is a bar graph showing a cell phenotype distribution according to an embodiment. FIG. 15 is a bar graph showing a comparison of various cytokine combinations and various administration methods according to one embodiment. FIG. 16 is a bar graph showing the number of viable cells at different circulation rates and different reactor operations for one embodiment. FIG. 17 is a bar graph showing the total number of harvested cells at various time points when culturing with a combination of different cytokines according to one embodiment. FIG. 18 is a bar graph showing cell yields when different activators are used according to one embodiment. FIG. 19 is a bar graph showing the cell phenotype when different activators are used according to one embodiment. FIG. 20 is a graph showing the production of cytokines according to one embodiment. FIG. 21 is a graph showing the production of cytokines according to other embodiments.

The principles of the present invention will be further understood by reference to the following detailed description and embodiments shown in the accompanying drawings. Although specific features are shown and described in the detailed embodiments, it should be understood that the invention is not limited to the embodiments described below.

In the following, the embodiments shown in the accompanying drawings will be referred to and described in detail. Wherever possible, the same reference numerals are used for the same or similar parts in the drawings and in the description below.

FIG. 1 shows a front view of an example of a hollow fiber bioreactor 100 used with the present invention. The hollow fiber bioreactor 100 has a longitudinal axis LA-LA and includes a chamber housing 104. In at least one embodiment, the chamber housing 104 has four openings or four ports, namely IC inlet port 108, IC exit port 120, EC inlet port 128, EC outlet port 132.

In the embodiments of the present disclosure, the fluid in the first circulation path enters the hollow fiber bioreactor 100 through the IC inlet port 108 at the first longitudinal end 112 of the hollow fiber bioreactor 100 and enters the hollow fiber bioreactor 100. The second of the hollow fiber type bioreactor 100, which enters and passes through the inside of the capillary tube of the hollow fiber 116 (referred to as “IC” side (“IC”) side or “IC space” of the hollow fiber membrane in various embodiments). It exits the hollow fiber bioreactor 100 through an IC outlet port 120 located at the longitudinal end 124. The fluid path between the IC inlet port 108 and the IC outlet port 120 defines the IC portion 126 of the hollow fiber bioreactor 100. The fluid in the second circulation path enters the hollow fiber type bioreactor 100 through the EC inlet port 128, and is outside or outside the capillary tube of the hollow fiber 116 (referred to as “EC side” or “EC space” of the membrane). Out of the hollow fiber bioreactor 100 through the EC outlet port 132. The fluid path between the EC inlet port 128 and the EC outlet port 132 constitutes the EC portion 136 of the hollow fiber bioreactor 100. The fluid that has entered the hollow fiber bioreactor 100 through the EC inlet port 128 contacts the outside of the hollow fiber 116. Small molecules (eg, ions, water, oxygen, lactic acid, etc.) can diffuse through the hollow yarn 116 from the inside of the hollow yarn, that is, the IC space to the outside, that is, the EC space, or from the EC space to the IC space. High molecular weight molecules such as growth factors are usually too large to pass through the hollow fibers and remain in the IC space of the hollow fibers 116. The medium may be replaced and replenished if necessary. Further, if necessary, the medium may be circulated through an oxygen supply device or a gas transfer module to exchange gas (for example, cell proliferation system 500 (FIG. 5) or cell proliferation system 600 (FIG. 6). reference). The cells can be contained in the first and / or second circulatory tracts and can be present on the IC side and / or EC side of the membrane, as described below.

The material used to make the hollow fiber membrane may be any biocompatible polymeric material that can be processed into hollow fibers. In the embodiments of the present disclosure, one material that can be used includes synthetic polysulfone-based materials. In order to attach the cells to the surface of the hollow fiber, the surface is modified by at least coating the cell growth surface with a protein such as fibronectin or collagen, or irradiating the surface with radiation. Good. By treating the surface of the membrane with gamma rays, it becomes possible to attach adhesive cells to the membrane without further coating with fibronectin or the like. Bioreactors made of gamma-ray treated membranes can be reused. In embodiments of the present disclosure, other coatings and / or other treatments for cell adhesion may be used.

FIG. 2 shows an embodiment of a cell proliferation system 200 having a pre-installed fluid transport assembly according to an embodiment of the present disclosure. CES200 has a cell proliferation device 202. The cell proliferation device 202 has a hatch or an openable / closable door 204 that engages the rear portion 206 of the cell proliferation device 202. The interior space 208 within the cell proliferation device 202 is characterized in that it can accept and engage and secure a pre-installed fluid transport assembly 210 that includes the bioreactor 100. The pre-installed fluid transport assembly 210 is detachably attached to the cell proliferation device 202 to replace the fluid transport assembly 210 used in the cell proliferation device 202 with a new or unused fluid transport assembly 210. It can be replaced relatively quickly. By the operation of a single cell proliferation device 202, the first fluid transport assembly 210 is used to grow or proliferate a first set of cells, after which the first fluid transport assembly 210 is subjected to a second fluid transport. A second set of cells can be grown or proliferated using the second fluid transport assembly 210 without sterilization upon replacement for the assembly 210. The pre-installed fluid transport assembly has a bioreactor 100 and an oxygen supply or gas transfer module 212. In an embodiment, a pipe guide slot for receiving various media pipes connected to the fluid transport assembly 210 is shown as 214.

FIG. 3 shows the posterior portion 206 of the cell proliferation device 202 prior to detachably attaching the pre-installed fluid transport assembly 210 (see FIG. 2) according to the embodiments of the present disclosure. The door 204 that can be opened and closed (see FIG. 2) is omitted in FIG. The posterior portion 206 of the cell proliferation device 202 is provided with a plurality of different structures that operate in combination with the components of the fluid transport assembly 210. Specifically, the rear portion 206 of the cell proliferation device 202 is a plurality of peristaltic pumps (IC circulation pump 218, EC circulation pump 220, IC inlet pump 222, EC inlet pump) that cooperate with a pump loop in the fluid transport assembly 210. 224). Further, the rear portion 206 of the cell proliferation device 202 has a plurality of valves (IC circulation valve 226, reagent valve 228, IC medium valve 230, air removal valve 232, cell inlet valve 234, wash valve 236, distribution valve 238, EC. It has a medium valve 240, an IC disposal valve 242, an EC disposal valve 244, and a harvesting valve 246). Further, a plurality of sensors (IC outlet pressure sensor 248, IC inlet pressure / temperature sensor 250, EC inlet pressure / temperature sensor 252, EC outlet pressure sensor 254) are associated with the rear portion 206 of the cell proliferation device 202. In addition, an optical sensor 256 for the air removal chamber is shown.

A shaft or rocker control unit 258 for rotating the bioreactor 100 according to the embodiment is shown in FIG. A shaft access opening (eg, piping) of the fluid transport assembly 210 or fluid transport assembly 400 to the posterior portion 206 of the cell proliferation device 202 by a shaft fitting section 260 associated with a shaft or rocker control section 258. The opening 424 (FIG. 4) of the storage portion 300 (FIG. 4) can be properly aligned. Rotation of the shaft or rocker control unit 258 imparts rotational motion to the shaft fitting unit 260 and the bioreactor 100. Thus, when an operator or user of the CES 200 attaches a new or unused fluid transport assembly 400 (FIG. 4) to the cell proliferation device 202, the alignment is such that the fluid transport assembly is relative to the shaft fitting 260. It is a relatively simple task of properly orienting the axial access opening 424 (FIG. 4) of the 210 or fluid transport assembly 400.

FIG. 4 is a perspective view of a pre-installed fluid transport assembly 400 that is removable and removable. The pre-attached fluid transport assembly 400 is detachably attached to the cell proliferation device 202, bringing the fluid transport assembly 400 used in the cell proliferation device 202 to a new or unused fluid transport assembly 400. It can be replaced relatively quickly. As shown in FIG. 4, the bioreactor 100 is attached to a bioreactor coupling that includes a shaft fitting portion 402. The shaft fitting 402 is one or more (such as a urged arm or spring member 404) for engaging the shaft of the cell proliferation device 202 (eg, shaft 258 shown in FIG. 3). It has a shaft fastening mechanism.

In an embodiment, the shaft fitting 402 and the spring member 404 are connected to the mechanism of a cell proliferation system that rotates the bioreactor 100. For example, in some embodiments, the cell proliferation system may be part of a Quantum® Cell Growth System (CES) (Lakewood, Colorado, TERUMO BCT) capable of rotating the bioreactor. Good. An example of a cell proliferation system capable of rotating a bioreactor is at least US Pat. No. 8,399,245, issued March 19, 2013 (Title: Rotation system for cell growth chamber of cell proliferation system). And how to use it), US Pat. No. 8,809,043 issued February 13, 2013 (Title: Rotational System for Cell Growth Chamber of Cell Proliferation System and How to Use It), and 6/2015 It is described in US Pat. No. 9,057,045 issued on 16th May (Title: Method of Cell Input and Dispersion in Bioreactor of Cell Proliferation System). The entire US patent application described above is expressly incorporated into this application by disclosure herein.

According to the embodiment, the fluid transport assembly 400 has pipes 408A, 408B, 408C, 408D, 408E and the like and various fittings. These provide fluid paths as shown in FIGS. 5-9, as described below. Pump loops 406A, 406B, 406C are also provided for the pump. Various media may be provided where the cell proliferation device 202 is located, but in embodiments, the premount type fluid transport assembly 400 is sufficient to extend outside the cell proliferation device 202. By having a pipe of various length, the pipe associated with the medium bag may be joined to the pipe.

FIG. 5 is a schematic diagram of an embodiment of the cell proliferation system 500. FIG. 6 is a schematic view of the cell proliferation system 600, which is another embodiment. In the embodiments shown in FIGS. 5 and 6, cells are grown in IC space, as described below. However, the present invention is not limited to such examples. In other embodiments, cells may grow in EC space.

FIG. 5 shows the CES 500 according to the embodiment. The CES 500 has a first fluid circulation path 502 (also referred to as "capillary inner loop" or "IC loop") and a second fluid circulation path 504 (also referred to as "capillary outer loop" or "EC loop"). The first fluid flow path 506 is fluidly associated with the hollow fiber bioreactor 501 and at least partially constitutes the first fluid circulation path 502. The fluid flows into the hollow fiber bioreactor 501 via the IC inlet port 501A, passes through the hollow fibers in the hollow fiber bioreactor 501, and flows out through the IC outlet port 501B. The pressure gauge 510 measures the pressure of the medium leaving the hollow fiber bioreactor 501. The medium flows through the IC circulation pump 512, which is used to control the medium flow rate / circulation flow rate. The IC circulation pump 512 is capable of pumping fluid in a first direction (eg, clockwise) or in a second direction (eg, counterclockwise) opposite to the first direction. The exit port 501B can be used as an inlet in the opposite direction. The medium flowing into the IC loop 502 flows in through the valve 514. As will be appreciated by those skilled in the art, additional valves and / or other devices may be placed in various locations to isolate the medium and / or characterize the medium along specific parts of the fluid pathway. It can also be measured. Therefore, it should be understood that the schematic shown shows one possible configuration for a plurality of elements of the CES 500 and is deformable within the scope of one or more embodiments.

For IC loop 502, media samples are obtained from sample port 516 or sample coil 518 during operation. The pressure / temperature measuring device 520 arranged in the first fluid circulation path 502 can measure the pressure and temperature of the medium during operation. The medium then returns to the IC inlet port 501A to complete the fluid circulation path 502. Cells grown / proliferated in the hollow fiber bioreactor 501 are flushed out of the hollow fiber bioreactor 501 and either enter the harvest bag 599 through valve 598 or are redistributed into the hollow fiber and further grown. This will be explained in detail below.

In the second fluid circulation path 504, the fluid enters the hollow thread bioreactor 501 via the EC inlet port 501C and leaves the hollow thread bioreactor 501 via the EC outlet port 501D. In the EC loop 504, the medium contacts the outside of the hollow fibers of the hollow fiber bioreactor 501, which allows the diffusion of small molecules into and from the hollow fibers.

Before the medium enters the EC space of the hollow filament bioreactor 501, the pressure and temperature of the medium can be measured by the pressure / temperature measuring device 524 arranged in the second fluid circulation path 504. After the medium has left the hollow filament bioreactor 501, the pressure of the medium in the second fluid circulation path 504 can be measured by the pressure measuring device 526. For EC loops, media samples are obtained from sample port 530 or from sample coils during operation.

In the embodiment, after leaving the EC outlet port 501D of the hollow yarn bioreactor 501, the fluid in the second fluid circulation path 504 passes through the EC circulation pump 528 to the oxygen supply or gas transfer module 532. The EC circulation pump 528 is capable of pumping fluid in both directions. The second fluid flow path 522 is fluidly associated with the oxygen supply or gas transfer module 532 via the oxygen supply inlet port 534 and the oxygen supply outlet port 536. During operation, the fluid medium flows into the oxygen supply or gas transfer module 532 via the oxygen supply inlet port 534 and outflows from the oxygen supply or gas transfer module 532 via the oxygen supply outlet port 536. To do. The oxygen supply or gas transfer module 532 adds oxygen to the medium in CES500 and removes air bubbles. In various embodiments, the medium in the second fluid circulation channel 504 is in equilibrium with the gas entering the oxygen supply or gas transfer module 532. The oxygen supply or gas transfer module 532 may be any oxygen supply or gas transfer device of suitable size. Air or gas flows into the oxygen supply or gas transfer module 532 through the filter 538 and out of the oxygen supply or gas transfer device 532 through the filter 540. Filters 538 and 540 reduce or prevent contamination of the oxygen supply or gas transfer module 532 and associated media. The air or gas purged from the CES 500 during part of the priming process can be vented to the atmosphere via an oxygen supply or gas transfer module 532.

In the configuration shown in the CES500, the fluid media in the first fluid circulation path 502 and the second fluid circulation path 504 flow through the hollow fiber bioreactor 501 in the same direction (in a parallel flow configuration). The CES500 may be configured to flow in a countercurrent.

In at least one embodiment, the medium containing the cells (from bag 562) and the fluid medium from container 546 are introduced into the first fluid circulation channel 502 via the first fluid flow path 506. The fluid vessel 562 (eg, a cell input bag or saline priming fluid for priming air out of the system) fluidly enters the first fluid flow path 506 and the first fluid circulation path 502 via a valve 564. Be associated.

The fluid vessel, ie media bag 544 (eg, reagent), is fluidly associated with the first fluid inlet 542 via valve 548, and the fluid vessel, ie media bag 546 (eg, IC medium) has valve 550. Fluidly associated with the first fluid inlet path 542 via, or the bag 544, 546 is fluidly associated with the second fluid inlet path 574 via valves 548, 550, 570. In addition, sterilized and sealable first and second input priming paths 508 and 509 are provided. The air removal chamber (ARC) 556 is fluidly associated with the first circulation path 502. The air removal chamber 556 may have one or more ultrasonic sensors. The sensors include upper and lower sensors for detecting air, lack of fluid, and / or gas / fluid boundaries, such as air / fluid boundaries, at specific measurement points within the air removal chamber 556. Is done. For example, near the bottom and / or top of the air removal chamber 556, ultrasonic sensors can be used to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, an optical sensor may be used according to an embodiment of the present disclosure. Air or gas purged from the CES 500 during part of the priming process or other protocols can be vented into the atmosphere from the air valve 560 through line 558 fluidly associated with the air removal chamber 556. Is.

EC medium (from bag 568) or wash solution (from bag 566) is added to the first or second fluid flow path. The fluid container 566 is fluidly associated with the valve 570. The valve 570 is fluidly associated with the first fluid circulation path 502 via the distribution valve 572 and the first fluid inlet path 542. Further, by opening the valve 570 and closing the distribution valve 572, the fluid container 566 can be fluidly associated with the second fluid circulation path 504 via the second fluid inlet path 574 and the EC inlet path 584. Similarly, the fluid container 568 is fluidly associated with the valve 576. The valve 576 is fluidly associated with the first fluid circulation path 502 via the first fluid inlet path 542 and the distribution valve 572. Further, by opening the valve 576 and closing the distribution valve 572, the fluid container 568 can be fluidly associated with the second fluid inlet path 574. As an option, a heat exchanger 552 may be provided for introducing the medium reagent or cleaning solution.

In the IC loop, the fluid is first pumped by the IC inlet pump 554. In the EC loop, the fluid is first pumped by the EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may be associated with an EC inlet path 584.

In at least one embodiment, the first and second fluid circulation paths 502, 504 are connected to a waste line 588. When the valve 590 is opened, the IC medium reaches the waste bag or outlet bag 586 via the waste line 588. Similarly, when valve 582 is opened, EC medium flows through the waste line 588 into the waste bag or outlet bag 586.

In an embodiment, cells are harvested via a cell harvest path 596. Cells from the hollow filament bioreactor 501 are harvested into the cell harvest bag 599 via the cell harvest path 596 and valve 598 by pumping the IC medium containing the cells.

Each component of the CES 500 is housed in a device or housing such as the cell proliferation device 202 (FIGS. 2 and 3), which maintains the cells and medium at a predetermined temperature.

FIG. 6 is a schematic diagram of another embodiment of the cell proliferation system 600. The CES 600 has a first fluid circulation path 602 (also referred to as "capillary inner loop" or "IC loop") and a second fluid circulation path 604 (also referred to as "capillary outer loop" or "EC loop"). The first fluid flow path 606 is fluidly associated with the hollow fiber bioreactor 601 to form the first fluid circulation path 602. The fluid flows into the hollow fiber type bioreactor 601 through the IC inlet port 601A, passes through the hollow fiber in the hollow fiber type bioreactor 601 and flows out through the IC outlet port 601B. The pressure sensor 610 measures the pressure of the medium leaving the hollow fiber bioreactor 601. In addition to pressure, in embodiments, the sensor 610 may be a temperature sensor that detects medium pressure and medium temperature during operation.

The medium flows through the IC circulation pump 612, which is used to control the medium flow rate or the circulation flow rate. The IC circulation pump 612 is capable of pumping fluid in a first direction (eg, counterclockwise) or in a second direction (clockwise) opposite to the first direction. The exit port 601B can be used as an inlet in the opposite direction. The medium flowing into the IC loop can flow through the valve 614. As will be appreciated by those skilled in the art, additional valves and / or other devices may be placed in various locations to isolate the medium and / or the properties of the medium along specific parts of the fluid pathway. Can also be measured. Medium samples are obtained from sample coil 618 during operation. The medium then returns to the IC inlet port 601A to complete the fluid circulation path 602.

Cells grown / proliferated in the hollow thread bioreactor 601 are flushed from the hollow thread bioreactor 601 and enter the harvest bag 699 via valve 698 and line 697. Alternatively, when valve 698 is closed, cells are redistributed into hollow fiber bioreactor 601 and further grown. The schematic shown shows one possible configuration for a plurality of elements of the CES 600, and it should be understood that modifications to the schematic are possible within the scope of one or more embodiments.

In the second fluid circulation path 604, the fluid enters the hollow yarn bioreactor 601 via the EC inlet port 601C and leaves the hollow yarn bioreactor 601 via the EC outlet port 601D. In the EC loop, the medium contacts the outside of the hollow fibers of the hollow fiber bioreactor 601, which, in one embodiment, allows the diffusion of small molecules into and from the hollow fibers within the chamber 601. Become.

Before the medium enters the EC space of the hollow thread bioreactor 601 the pressure and temperature of the medium can be measured by a pressure / temperature sensor 624 located in the second fluid circulation path 604. After the medium has left the hollow fiber bioreactor 601 the pressure and / or temperature of the medium in the second fluid circulation path 604 can be measured by the sensor 626. For EC loops, media samples are obtained from sample port 630 or from sample coils during operation.

After leaving the EC outlet port 601D of the hollow thread bioreactor 601 the fluid in the second fluid circulation path 604 passes through the EC circulation pump 628 to the oxygen supply or gas transfer module 632. In an embodiment, the EC circulation pump 628 is also capable of pumping fluid in both directions. The second fluid flow path 622 is fluidly associated with the oxygen supply or gas transfer module 632 via the inlet port 632A and the outlet port 632B of the oxygen supply or gas transfer module 632. During operation, the fluid medium flows into the oxygen supply or gas transfer module 632 via the inlet port 632A and out of the oxygen supply or gas transfer module 632 via the outlet port 632B. The oxygen supply or gas transfer module 632 adds oxygen to the medium in the CES 600 and removes air bubbles.

In various embodiments, the medium in the second fluid circulation channel 604 is in equilibrium with the gas entering the oxygen supply or gas transfer module 632. The oxygen supply or gas transfer module 632 may be any device of suitable size useful for oxygen supply or gas transfer. Air or gas flows into the oxygen supply or gas transfer module 632 through the filter 638 and out of the oxygen supply or gas transfer device 632 through the filter 640. Filters 638, 640 reduce or prevent contamination of the oxygen supply or gas transfer module 632 and associated media. The air or gas purged from the CES 600 during part of the priming process can be vented to the atmosphere via an oxygen supply or gas transfer module 632.

In the configuration shown as CES600, the fluid media in the first fluid circulation path 602 and the second fluid circulation path 604 flow through the hollow fiber bioreactor 601 in the same direction (in a parallel flow configuration). In certain embodiments, the CES 600 may be configured to flow in a countercurrent manner.

In at least one embodiment, the medium containing the cells (from a source such as a cell vessel, eg, a bag) is attached to attachment point 662 and the fluid medium from the medium source is attached to attachment point 646. The cells and medium are introduced into the first fluid circulation path 602 via the first fluid flow path 606. The attachment point 662 is fluidly associated with the first fluid flow path 606 via a valve 664. The attachment point 646 is fluidly associated with the first fluid flow path 606 via a valve 650. The reagent source may be fluidly connected to point 644 and associated with the fluid inlet path 642 via the valve 648, or may be associated with the second fluid inlet path 674 via the valves 648 and 672.

An air removal chamber (ARC) 656 is fluidly associated with the first circulation path 602. The air removal chamber 656 may have one or more sensors. The sensors include upper and lower sensors for detecting air, lack of fluid, and / or gas / fluid boundaries, such as air / fluid boundaries, at specific points of measurement within the air removal chamber 656. Is done. For example, near the bottom and / or top of the air removal chamber 656, ultrasonic sensors can be used to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, an optical sensor may be used according to an embodiment of the present disclosure. Air or gas purged from the CES 600 during part of the priming process or other protocols can be vented from the air valve 660 into the atmosphere through the line 658 fluidly associated with the air removal chamber 656. Is.

The EC medium source is attached to the EC medium attachment point 668. The cleaning solution source is attached to the cleaning solution attachment point 666. As a result, the EC medium and / or the washing liquid is added to the first fluid flow path or the second fluid flow path. The attachment point 666 is fluidly associated with the valve 670. The valve 670 is fluidly associated with the first fluid circulation path 602 via the valve 672 and the first fluid inlet path 642. Further, by opening the valve 670 and closing the valve 672, the attachment point 666 can be fluidly associated with the second fluid circulation path 604 via the second fluid inlet path 674 and the second fluid flow path 684. Similarly, the attachment point 668 is fluidly associated with the valve 676. The valve 676 is fluidly associated with the first fluid circulation path 602 via the first fluid inlet path 642 and the valve 672. Further, by opening the valve 676 and closing the distribution valve 672, the fluid container 668 can be fluidly associated with the second fluid inlet path 674.

In the IC loop, the fluid is first pumped by the IC inlet pump 654. In the EC loop, the fluid is first pumped by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with an EC inlet path 684.

In at least one embodiment, the first and second fluid circulation channels 602, 604 are connected to a waste line 688. When the valve 690 is opened, the IC medium passes through the disposal line 688 to the disposal bag or outlet bag 686. Similarly, when valve 692 is opened, EC medium flows into the waste bag or outlet bag 686.

After the cells are grown in the hollow fiber bioreactor 601 the cells are harvested via the cell harvest path 697. The cells from the hollow filament bioreactor 601 are harvested into the cell harvest bag 699 via the cell harvest path 697 by pumping the IC medium containing the cells with the valve 698 open.

Each component of the CES 600 is housed in a device or housing such as the cell proliferation device 202 (FIGS. 2 and 3), which maintains the cells and medium at a predetermined temperature. Further, in the embodiment, the parts of CES600 and CES500 (FIG. 5) may be combined. In other embodiments, the CES may include fewer or more parts than the parts shown in FIGS. 5 and 6 within the scope of the present disclosure. In embodiments, certain portions of CES500, CES600 are performed by one or more features of the Quantum® Cell Proliferation System (CES) manufactured by Termo BCT, Inc. (Lakewood, Colorado).

In certain embodiments with CES600, T cells are grown in CES600. In this embodiment, T cells are collected by a leukocyte apheresis process and introduced into bioreactor 601. T cells are introduced into bioreactor 601 and enter pathway 602.

In some embodiments, T cells are purified prior to introduction into bioreactor 601. Centrifuges and / or purification columns are used in the purification process. In the purification process, for example, LS columns and / or beads (eg, functional beads) from Miltenyi Biotec (Bergisch Gladbach, Germany) are used.

However, in certain embodiments, T cells may be added directly to bioreactor 601 after aggregation by the leukocyte apheresis process and without a purification process. In some embodiments, T cell growth is believed to be enhanced by introducing other cells into the bioreactor along with the T cells. Therefore, T cells may be unpurified and charged into the bioreactor along with other red blood cells, platelets, granulocytes, and / or white blood cells. In one embodiment, when unpurified T cells are charged, most of the T cells proliferate, resulting in more than about 90% or more than about 95% of the cells removed from the bioreactor after proliferation. An amount, or greater than about 98%, or greater than about 99%, is a T cell, the remaining cells being red blood cells, platelets, granulocytes, other white blood cells, and / or a combination thereof. In one embodiment, when unpurified T cells are introduced, most of the T cells proliferate, resulting in less than about 100%, or less than about 99%, or about 99% of the cells removed from the bioreactor after proliferation. Less than 98%, or less than about 97%, or less than about 96%, or less than about 95%, or less than about 94%, or less than about 93%, or less than about 92%, or less than about 91%, or about 90% Less than are T cells.

In some embodiments, T cells are added to bioreactor 601 after the priming step. As is understood, some T cells are non-adhesive and therefore do not need to adhere to the hollow fiber wall of bioreactor 601 for expansion / proliferation. In these embodiments, it is not necessary to coat the inside of the hollow fiber with a coating agent (eg, fibronectin) to promote adhesion. In this case, the (purified or unpurified) T cells are introduced into the bioreactor 601 after the priming step without the bioreactor coating step.

Upon entering the bioreactor 601 cells are exposed to activators to activate cell proliferation. In one example, antigen presenting cells are pre-introduced into bioreactor 601 or pre-grown in bioreactor 601. In one embodiment, monocyte-derived dendritic cells are grown in bioreactor 601 prior to adding T cells. In some embodiments, when antigen-presenting cells are used, the growth process is first performed under conditions optimized for the proliferation of antigen-presenting cells.

In other embodiments, antibody-functionalized beads are added to bioreactor 601. In other embodiments, such beads are added to a container containing the cells (eg, a bag containing T cells) before the cells are introduced into the bioreactor. Antibodies activate T cell proliferation. Examples of beads used include DYNABEADS, which are beads manufactured by Thermo Fisher Scientific (Waltham, Mass.). In some embodiments, the beads are surface-functionalized by the antibody. Examples of such antibodies include, but are not limited to, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD28 antibody, anti-CD34 antibody and the like.

In other embodiments, the activator takes the form of a fluid (eg, a solution). The activator-containing fluid comprises one or more activators (eg, antigen). In addition to the antigen, the fluid may contain other components. Examples include, but are not limited to, proteins, surfactants, reagents, buffers and the like. An example of a fluid containing a soluble co-stimulatory signal is IMMUNOCULT ™ human CD3 / CD28 T cell activator (STEMCELL Technologies, Canada).

After the cells have been exposed to the activator (and during continued exposure), the cells proliferate in bioreactor 601. During growth, a large number of substances are added to or removed from bioreactor 601. For example, cytokines are used to stimulate T cell proliferation. In some embodiments, a single cytokine is used. The cytokines used are not limited to the following, but are, for example, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7). , Interleukin 15 (IL-15), and combinations thereof. In other embodiments, only IL-2 is used. Alternatively, IL-2 is used with IL-7. Alternatively, only IL-7 is used. Alternatively, IL-7 is used with IL-15. In other embodiments, IL-2 is used with IL-15. In yet another embodiment, IL-2, IL-7 and IL-15 are used together. In some embodiments, when more than one cytokine is used, the cytokines may be added simultaneously, at different times, or in combination.

In some embodiments, the cytokine can also be added directly to the bioreactor 601 via, for example, port 618. However, in other embodiments, the cytokine is added, eg, via pathway 606, where the cytokine perfuses within the bioreactor 601. In some embodiments, perfusion of cytokines into the bioreactor 601 is believed to promote T cell growth.

In addition to stimulating T cells, T cells may be fed, for example, by adding a medium containing a large number of nutrients. In some embodiments, the medium is a commercially available medium. For example, the medium used to feed T cells is based on the TexMACS medium commercially available from Miltenyi Biotec (Bergisch Gladbach, Germany) (partially modified fluid produced). It may be the one that has been made, or it may be left as it is without any change). In other embodiments, the medium is based on the CELLGRO medium commercially available from Celgenic (Freiburg, Germany) (the produced fluid may be partially modified or intact without modification). Good). The medium may be partially modified by adding other substances (eg, salts, sera, proteins, etc.).

As part of T cell proliferation, other conditions such as temperature, pH, oxygen concentration, carbon dioxide concentration, excretion concentration, metabolite concentration, etc. are regulated within the bioreactor 601. In some embodiments, the flow rate on the EC side (eg, path 604) is used to control various parameters. For example, when it is desired to reduce the concentration of excreta or metabolite on the IC side where cell growth is performed, by increasing the flow rate on the EC side, the excrement is moved from the IC side through the hollow thread to the EC side. And / or metabolites can be reliably removed from the IC side.

After growing the T cells, the cells are removed from bioreactor 601. T cells are collected in container 699.

Embodiments of the invention have advantages (in T cell growth) over conventional processes by using CES600. For example, the use of hollow fibers allows for close cell-cell communication, thus increasing activation and stimulation of T cells and initiating and continuing proliferation. Also, by using a hollow filament bioreactor such as bioreactor 601 it is possible to provide a large surface area for T cell growth, resulting in higher concentrations of T cells or higher amounts of T cells. It is produced.

Further, the conditions (including the IC flow rate and the EC flow rate) in the bioreactor 601 can be controlled by using a large number of parts / members of the CES 600. Further, in CES600, cytokines can be introduced at various places, and cytokines can be introduced directly or indirectly (for example, perfusion) into bioreactor 601.

In addition, the CES 600 provides a closed system. That is, the steps for growing T cells can be performed without direct exposure to the surrounding environment. Therefore, the surrounding environment does not contaminate T cells, or the surrounding environment is not contaminated by T cells or substances used for T cell growth. In some embodiments, it is believed that the starting concentration of T cells for proliferation can be reduced to a smaller amount compared to other methods / systems. In these embodiments, the yield of T cell proliferation can be increased compared to other methods / systems.

In some embodiments, the time to grow an effective amount of T cells is believed to be reduced. In yet other embodiments, it is believed that T cells can grow with reduced use of substances such as (expensive) cytokines as compared to other methods / systems.

FIG. 7 shows a flow chart 700 of cell growth according to the embodiment. In the following, specific devices for performing the steps of Flowchart 700 will be described, but the present invention is not limited thereto. For example, some steps may be described as being performed by a part of the cell proliferation system or by a processor capable of performing the steps based on software given as a processor executable instruction. These are performed only for the purpose of illustration, and the flowchart 700 is not limited to being performed by a specific device.

Flowchart 700 begins at step 704 and proceeds to step 708 as an option to collect cells. As an example, step 708 may include an apheresis process. In one embodiment, the leukocyte apheresis process is performed as part of step 708. A device capable of collecting cells includes the COBE® Spectra Apheresis System manufactured by Termo BCT (Lakewood, Colorado).

Flow chart 700 proceeds from step 708 to step 712. In step 712, cells are introduced into the cell proliferation system, particularly the bioreactor of the cell proliferation system. As mentioned above, in some embodiments, flowchart 700 may start at step 712. In an embodiment, the bioreactor is a hollow fiber bioreactor such as Bioreactor 100 (see FIG. 1). In these embodiments, in step 712, cells may be individually flowed into one or more hollow fibers. In step 712, cells may be introduced into the bioreactor using a processor, pump, valve, fluid path, or the like. In one embodiment, step 712 may open valves (eg, valves 564, 514, 664, 614) and operate pumps (eg, pumps 554, 654).

After step 712, the flowchart 700 proceeds to step 716. In step 716, the cells are exposed to the activator. As will be appreciated, certain cell types require, for example, activation by exposure to certain proteins before they can be activated for proliferation. An example of this cell type is T cells. In step 716, the cells are exposed to the activators needed to activate the cells in order to proliferate and grow.

Step 716 may include a plurality of steps performed as part of step 716 or prior to step 716. For example, in some embodiments, step 720 is performed to grow antigen-presenting cells that present the activators needed to activate cell proliferation. In one embodiment, step 720 may be performed before step 716 or even before step 708. In one example, antigen presenting cells (eg, dendritic cells) are grown. In these embodiments, step 720 may include multiple steps (eg, collecting monocytes as progenitor cells to the antigen presenting cells) that result in the antigen presenting cells being present in the bioreactor. Antigen-presenting cells have a major histocompatibility complex (MHC) on the surface, and on the surface of antigen-presenting cells, an antigen that induces proliferation of the cells when presented to other cells (for example, T cells). Exists.

In other embodiments, step 716 may include step 724. In step 724, the beads are added to the bioreactor. As will be appreciated, in some embodiments, the beads (eg, magnetic beads, polymer beads, glass beads, ceramic beads) contain specific activators on the bead surface that activate cell proliferation. As such, it may be partially modified. As an example, magnetic beads are functionalized by one or more antibodies. Examples of antibodies include anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD28 antibody, and anti-CD34 antibody. When a cell is exposed to an antibody, it is activated to proliferate in the presence of other substances. Suitable beads for use in some embodiments include DYNABEADS®, which is a bead manufactured by Thermo Fisher Scientific (Waltham, Mass.).

After step 716, the process proceeds to step 728. In step 728, cells are proliferated. Step 728 may have a plurality of substeps. For example, in substep 732, cells are stimulated, for example, with cytokines. Such stimulation allows the cell proliferation to continue.

In embodiments, step 728 is performed over a period of hours, days, or weeks. During this period, various substeps (eg, 732, 736, and / or 740) may be performed at various time points. For example, during this period, substances such as cytokines (eg, interleukins) may be added and dispersed (perfused) or directly charged into the bioreactor to continue stimulating cell proliferation. Non-limiting examples of the cytokines used are interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 1. Leukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-) 10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Interleukin 18 (IL-18), Interleukin 23 (IL-23) and combinations thereof can be mentioned. Cytokines are used individually or in combination, for example, in one embodiment IL-7 is used in combination with IL-15. In other embodiments, IL-2 and IL-7 are used together. In yet other embodiments, IL-2 and IL-15 are used together.

Step 728 may include cell feeding step 736. In step 736, various nutrients may be added, including glucose, phosphates, salts and the like. Step 736 may include a step in which the nutrient concentration is detected and the nutrient is added to the bioreactor in response to a relatively low concentration measurement. Step 736 may include a step of performing cell feeding by adding nutrients using a processor, pump, valve, fluid path, or the like. Alternatively, the addition of nutrients such as glucose may be carried out according to a predetermined schedule.

Step 740 may include controlling the growth environment of the bioreactor. As will be understood, in addition to nutrients, there are several different parameters for the environment for optimizing cell growth. For example, temperature, pH, oxygen concentration, carbon dioxide concentration, emission concentration, biotransformation concentration and the like are monitored and controlled as part of step 740. In one embodiment, it flows through the capillary outer space (EC) side of the bioreactor in controlling pH, oxygen concentration, carbon dioxide concentration, excretion concentration, metabolite concentration, etc. in the capillary inner space where cell proliferation is performed. The flow rate of the fluid is used.

Flowchart 700 proceeds from step 740 to step 744 for removing cells from the bioreactor. Step 744 may include a plurality of substeps. For example, the harvesting process itself involves multiple steps, but can be done as part of step 744. In embodiments, step 744 may include changing the circulation rates on the inner space (IC) and EC sides of the capillaries of the bioreactor. In another embodiment, step 744 may include a step of circulating various substances to ensure that cells attached to the inner surface of the hollow fiber are detached and removed from the bioreactor. As an example, the addition of proteases degrades proteins such as glycoproteins that help cells bind to hollow fibers. Flowchart 700 ends in step 752.

According to the embodiments of the present disclosure, the operation steps described in the flowchart shown in FIG. 7 are for illustrative purposes only and may be modified, combined with other steps, or with other steps. It may be done in parallel. In embodiments, steps may be reduced or steps may be added as long as they do not deviate from the scope of the present disclosure. Also, for example, the steps (and sub-steps) may be performed automatically in some embodiments, for example, by a processor performing a pre-programmed task stored in memory. Here, such steps are presented for illustrative purposes only.

Although various steps have been described by enumerating them in a specific order with respect to the flowchart 700, the present disclosure is not limited to such an order. In other embodiments, the steps and substeps may be performed in different orders, in parallel, or in part in the order shown in FIG. 7, or in the sequence shown in FIG. Also, although steps or substeps have been described as being performed with specific features or specific structures, this is not intended to limit the invention. Rather, these descriptions are for illustrative purposes only. In other embodiments, one or more of the steps in Flowchart 700 may be performed using other structures and features not described above. Further, the flowchart 700 may include an optional step. However, the steps described above, which are not shown as options, should not be considered essential in the present invention and may be performed in some embodiments and not in other embodiments.

FIG. 8 shows an example of components of a computer system 800 in which an embodiment of the present invention is executed. The computer system 800 may include, for example, a task in which the cell proliferation system uses a processor (such as a custom task or a pre-programmed task performed as part of a process, such as the process shown in Flowchart 700 above). Is used in embodiments that carry out.

The computer system 800 has a user interface 802, a processing system 804, and / or a storage device 806. The user interface 802 includes an output device 808 and / or an input device 810, as will be appreciated by those skilled in the art. The output device 808 may have one or more touch screens. The touch screen may have a display area for providing one or more application windows. The touch screen may also be, for example, an input device 810 capable of accepting and / or capturing physical contact from a user or operator. The touch screen may be a capacitive liquid crystal display (LCD) that allows the processing system 804 to estimate the contact position, as will be appreciated by those skilled in the art. In this case, the processing system 804 can copy the contact position to a user interface (UI) element displayed at a predetermined position in the application window. The touch screen can also accept contact via one or more other electronic structures according to the present invention. Examples of the other output device 808 include a printer, a speaker, and the like. Examples of the other input device 810 include a keyboard, other touch-type input devices, a mouse, a voice input device, and the like, as can be understood by those skilled in the art.

In an embodiment of the invention, the processing system 804 may have a processing unit 812 and / or a memory 814. The processing unit 812 may be a general-purpose processor capable of operating to execute an instruction stored in the memory 814. The processing unit 812 may include a single processor or a plurality of processors in the embodiment of the present invention. Further, in the embodiment, each processor may be a multi-core processor having one or more cores for individually reading and executing instructions. Processors may include general purpose processors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and other integrated circuits, as will be appreciated by those skilled in the art.

In embodiments of the invention, memory 814 may include any storage device that stores data and / or processor executable instructions in the short or long term. The memory 814 is, for example, a random access memory (RAM), a read-only memory (ROM), or an electrically erasable, programmable read-only memory (EEPROM), as can be understood by those skilled in the art. including. Other storage media include, for example, CD-ROMs, tapes, digital versatile discs (DVDs), or other optical storage devices, tapes, magnetic disk storage devices, magnetic tapes, as can be understood by those skilled in the art. , Other magnetic storage devices, etc.

The storage device 806 is any long-term data storage device or component. In embodiments of the present invention, storage device 806 may include one or more of the systems described in connection with memory 814. The storage device 806 may be permanently installed or removable. In one embodiment, the storage device 806 stores data generated or given by the processing system 804.

Examples Some examples of methods / processes / protocols for implementing embodiments are described below. Specific features (cell type, cytokines, antigen presentation, etc.) are described in these examples, but they are for illustrative purposes only. The present invention is not limited to the following examples.

Example 1
To generate a baseline for comparison of the Quantum® Cell Proliferation System (CES), monocytes purified from leukocyte apheresis products were matured into dendritic cells and Wolfl (o in Wolfl). Umralt is actually attached above, but it is omitted because it is a character that cannot be used in electronic applications) and according to the Greenberg protocol (Nature Protocols Vol. 9, No. 4, 2014) (preserved cryopreserved from leukocyte apheresis products). T) Donor-matched T cells (combined). The entire protocol is expressly incorporated into this application by disclosure herein.

One donor cell was cultured for 23 days (5 days DC maturation and 18 days co-culture) to determine the optimal culture period. Cell proliferation was observed to continue until day 20 of cell culture. After the 20th day, the number of cells and the viability decreased as shown in FIGS. 9 and 10.

The donor 2 was used to determine the optimum monocyte purity and optimum seeding density. Monocytes were sown on 6-well culture plates. One is about 50% pure seeded directly from the fifth fraction in the elution process, and the other is magnetic bead purification performed through two LS columns (Miltenny) using CD14 microbeads. It is of the later 99% purity. Under the above two cell purity conditions, the cells were seeded at a density of 10E + 06 cell count / mL, sequentially diluted 2-fold and up to 3.13E + 05 cell count / mL. Optimal seeding densities for purified monocytes were observed at 2.5E + 06 cell count / mL and 1.25E + 06 for unpurified cells (see Figure 11).

Monocyte-derived dendritic cells were pooled under conditions prior to co-culture with T cells, and T cells were co-cultured with monocyte-derived dendritic cells at a ratio of 4: 1 for 19 days. It was observed that cells seeded directly from the eltriation process performed T cell proliferation as well, if not more, than purified monocytes (see Figure 12). Similar to donor 1, cell viability decreases as cell proliferation levels off (see Figure 13).

Example 2
T cells proliferate well in QUANTUM® CES by the following two measures. That is, co-stimulation of CD3 and CD28 with antibody-functionalized paramagnetic beads (DynaBeads T cell Activator) (Q1070), and professional antigen presentation matured from monocytes present in the original leukocyte aferesis product. It is a measure by antigen presentation by monocyte-derived dendritic cells (DCs) (Q1069) which are cells (APCs). These two measures exert an initial effect on the proliferation of T cell cultures in QUANTUM® CES (see FIG. 14).

FIG. 15 shows the phenotypic distribution of cells proliferated in the QUANTUM system for proof-of-concept. In both DC-stimulated proliferation and bead-stimulated proliferation, a shift was observed in the phenotypic distribution (CD4: CD8 ratio) for both the starting product and the cells grown in the T175 flask. This initial data suggests that QUANTUM (registered) CES favors CD8 T cell proliferation regardless of the stimulation method.

Table 1 shows the number of cells input and the number of harvested cells in the proliferation for proof of concept.

Example 3
Many cytokines promote T cell proliferation. Examples of such cytokines include IL-2 and a combination of IL-7 and IL-15. As a method for administering these cytokines, there are two candidates, a bolus injection method and a continuous perfusion method of a pre-configured medium containing an appropriate cytokine concentration. This was investigated by a study of 7 groups. In this study, IL-2 alone, a combination of IL-7 and IL-15, and a combination of all three cytokines were fed to QUANTUM® CES by the above two methods and compared directly. The control group in this study is a group that does not input cytokines. From this study, it was found that a higher magnification of proliferation was obtained in the continuous perfusion method of the combination of IL-7 and IL-15 (see FIG. 15). FIG. 15 shows the number of harvests in the growth of QUANTUM® CES performed for comparison with various combinations of cytokines and two administration methods.

Example 4
The next step is to examine the increase in IC circulation rate during cell proliferation and the addition of 6 degree swing to the bioreactor. The application of these new conditions to the culture environment was found to have a negative effect on T cell proliferation (see Figure 16). FIG. 16 shows a comparison of the number of harvests between the control group (bioreactor stationary, circulation rate 5 ml / min) and the group that increases the IC circulation rate while swinging (± 6 degree swing, circulation rate 20 ml / min). It is a graph.

Example 5
T cell proliferation is supplemented with recombinant human interleukin 2 (improved sequence) (IL-2 IS) (Miltenyi Biotec, Germany) and recombinant human interleukin 7 (IL-7) (Miltenyi Biotec, Germany). Promoted with TexMACS GMP grade medium (Miltenyi Biotec, Germany). T cell proliferation is a bound co-stimulatory signal, ie DynaBeads® Human T-Activator CD3 / CD28 (Life Technologies, Grand Island, NY) or soluble co-stimulatory signal or ImmunoCult ™ Human. It is initiated using the CD3 / CD28 T Cell Activator (STEMCELL Technologies, NY). Quantum® CES (Thermo BCT, Lakewood, Colorado) is primed with Phosphate Buffered Saline (PBS) (Lonza, Switzerland) to remove air from the system. According to the priming sequence, PBS is exchanged for TeXMACS GMP medium.

Fresh and normal peripheral blood leukocyte apheresis preparation (LeukoPak) is a single donor (AllCells, Alameda, CA) using the COBE® Spectra Apheresis System (Terumo BCT, Lakewood, Colorado). And from Hemacea, Van Knys, Calif.). Upon arrival in the lab, cell concentration and viability will be measured by the Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA). In addition, the percentage of lymphocytes is measured by a Coulter Ac-T diff2 (Beckman Coulter, Brea, Calif.) Blood analyzer.

Lymphocytes aliquot from leukapheresis preparations 1.0 × 10 ⁸ is, cell: bead ratio of 2: 1 and DynaBeads (R) in or at 2.5μl against 1.0 × 10 ⁶ lymphocytes Combined with ImmunoCult ™, and further combined with 1.0 × 10 ⁵ IU IL-2 and 2.5 × 10 ³ ng IL-7, 100 mL volume in TexMACS GMP medium in sterile storage bottles. Be made. The cell / cytokine / stimulus vector solution is processed and placed in Cell Inlet Bag (Terumo BCT, Lakewood, Colorado) and charged into the IC side of the bioreactor while circulating the medium, and the solution is uniformly distributed in the hollow thread. Be distributed.

After cell transfer, the TexMACS medium was circulated at 1 mL / min on the IC side and 100 mL / min on the EC side, and a continuous medium containing IL-2 (200 IU / mL) and IL-7 (5 ng / mL) was squeezed. Flow is performed at 0.1 mL / min at the IC inlet of the bioreactor. The bioreactor orientation is stationary at 0 ° for the first 6 days of growth except days 3, 6, and 9. On the 3rd, 6th and 9th days, the IC circulation and the EC circulation were set to 100 mL / min, respectively, and 150 mL of medium containing IL-2 and IL-7 (DynaBeads® proliferation) was administered by bolus or. Bolus administration of 150 mL (ImmunoCult ™ growth) of medium containing IL-2 and IL-7 and soluble costimulatory signal was performed at 30 mL / min on the IC side of the bioreactor, and the bioreactor was paused for 1 second. Continuously swing between -90 ° and 180 °. Upon completion of bolus administration, the bioreactor is returned to a resting 0 ° state and the inlet and circulation pump settings are returned to growth initiation values.

From day 6, the bioreactor is flipped 180 ° between 0 ° and 180 ° every 24 hours. The IL-2 concentration and IL-7 concentration in bolus administration are changed for each administration (see Table 2). After each bolus administration, the cells were in a uniform suspension and part of the IC sample loop was cleaved and joined using a TSCD® II aseptic joining device (Terumo BCT, Lakewood, Colorado). To. This sample is used to monitor cell concentration, viability, cell phenotype and secretory cytokines over the growth period.

Cells are harvested from QUANTUM® CES on days 7, 10, or 12 of proliferation using a High Density Washout automated task. To facilitate this harvesting method, the medium ran a bioreactor (continuous oscillation between -90 ° and 180 ° with a 1 second pause) to achieve a well-mixed cell suspension. However, on the IC side, the cells are circulated at a rate of 100 mL / min for 5 minutes. After media circulation, the harvest bag is joined to the disposal line instead of the disposal bag. The task settings for high density washout are: IC inlet = 200 mL / min, EC circulation rate = 300 mL / min and stop condition = 1000 mL. Cytokine-free medium is used for this task. After harvest, cell viability, absolute number, and phenotypic properties are analyzed by flow cytometry.

Cells are characterized as T cells by the expression of CD3. In addition, the helper subset and the cytotoxic subset are characterized by the expression of CD4 and CD8, respectively. Both surface and intracellular receptors on CD3 in the sample are colored using UCHT1 clones for CD3 associated with PerPC-Cy5.5 (BD Biosciences, San Jose, CA). SK3 clones of CD4 linked to PE (BD Biosciences, San Jose, CA) are used to color the surface receptors on CD4 in the sample. BW135 / 80 clones of CD8 associated with BioBright FITC (Miltenny Biotec, Germany) are used to color the surface receptors on CD8 in the sample. Non-viable cells were distinguished from the analysis using an eFluor® 780 cell viability dye (eBioscience, San Diego, CA). Cell surface and intracellular coloring was performed using the FIX & PERM® Cell Permeabilization Kit (Life Technologies, Grand Island, NY). Samples were analyzed in FACSCanto II (BD Biosciences, San Jose, CA) using FACSDiva software version 6.1.3. (BD Biosciences, San Jose, CA). FACSCanto II was calibrated by BD FACSDiva CS & T Beads (BD Biosciences, San Jose, Calif.) And the correction amount was BD CompBeads (BD Biosciences, San Jose, Calif.) And ArC ™ amine-reactive dyes. Calculated using a Compensation Beads Kit (Amine Reactive Compensation Bed Kit) (Life Technologies, Grand Island, NY). 10,000 events were recorded for one sample.

Samples were periodically removed from the IC and EC sides of the bioreactor during maturation and stored at -20 ° C. The samples were then warmed to room temperature and used with the Luminex ™ Assay (Assay) Th1 / Th2 / Th17 Human Magic 8-Plex Panel (Life Technologies, Grand Island, NY), IL-2, IL. -4, IL-5, IL-9, IL-10, IL-13, IL-17, IFNγ were analyzed. The analyte was analyzed by running xPONENT (Luminex, Austin, Texas) collection and analysis software using a MAGPIX analyzer (Luminex, Austin, Texas).

It can be seen that the use of IL-7 with IL-2 in QUANTUM® CES significantly increases T cell proliferation as compared to the use of IL-2 alone (see FIG. 17). In QUANTUM® CES, T cell proliferation with bead-bound CD3 and CD28 co-stimulatory antibodies is superior to proliferation of soluble costimulatory antibodies (see FIG. 18). Proliferation products after 7 days were consistently about 95% CD3 + cells in bead growth, with the majority of solubility-based growth being in similar percentages (see Figure 19). However, the viability of cell products after growth is consistently better with beaded growth than with corresponding soluble-based growth for all donors (see Table 3 below). In Table 3, *** indicates that the growth was unmeasurable due to system contamination. ** indicates that the analysis was performed on the corresponding paired growth. Cytokine analysis in all experiments shows an increase in cytokines associated with Th1 and cytotoxic subsets with an increase in IL-9. It has been known that IL-9 promotes cell proliferation and suppresses natural cell death by stimulating the IL-2 receptor group by adding IL-7 to the culture medium (FIGS. 20 and 21). ..

Quantum® CES can proliferate more than 500-fold T cells from a fraction of apheresis blood collection. Manufacturing process control of automated and functionally closed systems can reduce workload and reduce open events in T cell maturation. As a result, the production of cell products can be performed more efficiently.

Those skilled in the art will appreciate that various modifications and modifications can be made to the methods and structures of the present invention without departing from the scope of the present invention. Therefore, it should be understood that the present invention is not limited to the particular embodiments or examples described above. Rather, the invention is intended to include variants and modifications within the scope of the following claims and their equivalents.

Although embodiments and applications of the present invention have been shown and described, it should be understood that the present invention is not limited to the configurations described above. Various modifications, changes, etc. apparent to those skilled in the art can be made in detail in the configuration, operation, method and system of the present invention without departing from the scope of the present invention.

Claims (12)

Using the leukocyte apheresis process, a step of producing a first plurality of cells containing leukocytes and some of which are T cells,
Said first plurality of cells, comprising the steps of introducing into the hollow-fiber bioreactor having a plurality of hollow fibers,
A step of exposing the first plurality of cells to an activator to activate the proliferation of the first plurality of cells in the hollow fiber bioreactor.
A step of proliferating at least a part of the first plurality of cells in the plurality of hollow fibers of the hollow fiber type bioreactor to generate a second plurality of proliferating cells.
A step of removing the second plurality of proliferating cells from the bioreactor,
Have,
In the step of introducing the first plurality of cells into the hollow fiber type bioreactor, the first plurality of cells are generated by the leukocyte apheresis process and then directly introduced into the bioreactor without undergoing a purification process. ,
How to grow cells. The method for growing cells according to claim 1 , wherein the first plurality of cells are added to the hollow fiber bioreactor without additional purification treatment. The method for proliferating cells according to claim 2 , wherein the first plurality of cells contain erythrocytes. The method for proliferating cells according to claim 3 , wherein the first plurality of cells contain platelets. The method for proliferating cells according to claim 4 , wherein the first plurality of cells contain granulocytes. In the method for growing cells according to claim 5 , the exposure of the first plurality of cells to the activator exposes the first plurality of cells to beads having the activator on the surface. A method of cell proliferation, characterized in that it comprises. In the method for proliferating cells according to claim 6 , the exposure of the first plurality of cells to the activator exposes the first plurality of cells to dendritic cells having the activator. A method of cell proliferation, characterized in that it comprises. The method for growing cells according to claim 7 , wherein the activator contains one or more antigens. In the method for growing cells according to claim 6 , the activator comprises one or a plurality of antibodies selected from the group consisting of anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody and a combination thereof. A characteristic method of cell proliferation. The method for growing cells according to claim 1, wherein the step is performed in a closed system. In the method for proliferating cells according to claim 9, the step of proliferating at least a part of the first plurality of cells to generate a second plurality of proliferating cells is the hollow fiber bioreactor. A method of cell proliferation comprising the step of circulating a medium in a reactor. In the method for growing cells according to claim 11 , the medium is interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7). , Interleukin 15 (IL-15), and one or more of combinations thereof.